![Characterization of KLF1+/E325K (CDA IV) cell line. (A) Images of WT and KLF1+/E325K cells stained with May-Grünwald Giemsa. Original magnification ×400-800. Scale bars, 20 μm. Orange arrowheads indicate multinuclear proerythroblasts, red arrowheads indicate cells with membrane blebbing, and blue arrowheads indicate chromatin bridges. (B) Quantification of western blots (see supplemental Figure 1G; mean ± standard deviation [SD]; n = 3; ∗P < .05) for WT, KLF1+/E325K, and KLF1+/− expanding cells probed with KLF1 antibody, with β-actin as the loading control. (C) Proportion of multinuclear cells in expanding cell population quantified as a percentage of total live cells (n = 5; >100 cells counted per sample; ∗∗P < .01). (D) Cumulative fold expansion and (E) viability of WT, KLF1+/E325K, KLF1+/−, and KLF1+/ΔE325K cells during expansion (day 0) and on days 2, 4, 6, 8, 10, and 12 of differentiation (mean ± SD; n = 11. ∗P < .05 and ∗∗∗P < .001 for WT vs KLF1+/E325K; +P < .05 and +++P < .001 for WT vs KLF1+/−). (F) Percentage of cells tested positive for anV with fluorescein isothiocyanate (FITC) and negative with propidium iodide (PI) during expansion (day 0) and on days 2, 4, 6, 9, and 11 of differentiation (mean ± SD; n = 3; ∗∗∗P < .001). (G) Western blot of WT, KLF1+/E325K, and KLF1+/− cells probed with antibody to cleaved caspase 3, and to β-actin as loading control (representative of 3 biological repeats). (H) Percentage of cells positive for anV using FITC and PI during expansion (day 0) and on days 2, 4, 6, 9, and 11 of differentiation (mean ± SD; n = 3; ∗∗∗P < .001). (I) Percentage of erythroid cell types during expansion (day 0) and on days 2, 4, 6, 8, and 10 of differentiation, analyzed after staining with May-Grünwald Giemsa (mean ± SD; n = 5; >100 cells counted per sample). (J) Expression of (i) BCAM, (ii) AQP1, and (iii) ICAM4 determined via quantitative polymerase chain reaction (qPCR; mean ± SD; n = 4; ∗P < .05; ∗∗∗P < .001), and abundance of (iv) CD44 protein determined via flow cytometry using an allophycocyanin (APC)-labeled anti-CD44 antibody (mean ± SD; n = 3; ∗P < .05; ∗∗∗P < .001). BasoE, basophilic erythroblast; Ortho, orthochromatic erythroblast; ProE, proerythroblast; Poly, polychromatic erythroblast; Retic, reticulocyte.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/141/25/10.1182_blood.2022018735/3/blood_bld-2022-018735-gr1.jpeg?Expires=1725146033&Signature=lSfvNs8LDRnaapqnmbpJX~kez2yh2w7EU1SsysBWT4bBZk7IKPpt0bXZGz4qlzLBzucZ~5fNZyYQKtEchtyGq2D3mBYxpmtSO81EwKn3fWYx1pCIczGExHqBqHCdwWWgbbr9RNI8MhxM~YutAiGZ6TvZWRk6VTzEw236IhcS4udUke-nrkWPEt~jQeE5-dzN8SBcIhi6U77q7EupNA7G4xuaU5Vzu4KtASjUY4EErIJ-oYN37q-QFFnnxZrUOHcJAVdgG3d5GzN~TkfpBLJxsI3rajvQ6Z6mXgoKDujNiK6yJtWP1FcrrXx7qfIaJp4StU6rqKlFacoQSYbAGVnxIQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Characterization of KLF1+/E325K (CDA IV) cell line. (A) Images of WT and KLF1+/E325K cells stained with May-Grünwald Giemsa. Original magnification ×400-800. Scale bars, 20 μm. Orange arrowheads indicate multinuclear proerythroblasts, red arrowheads indicate cells with membrane blebbing, and blue arrowheads indicate chromatin bridges. (B) Quantification of western blots (see supplemental Figure 1G; mean ± standard deviation [SD]; n = 3; ∗P < .05) for WT, KLF1+/E325K, and KLF1+/− expanding cells probed with KLF1 antibody, with β-actin as the loading control. (C) Proportion of multinuclear cells in expanding cell population quantified as a percentage of total live cells (n = 5; >100 cells counted per sample; ∗∗P < .01). (D) Cumulative fold expansion and (E) viability of WT, KLF1+/E325K, KLF1+/−, and KLF1+/ΔE325K cells during expansion (day 0) and on days 2, 4, 6, 8, 10, and 12 of differentiation (mean ± SD; n = 11. ∗P < .05 and ∗∗∗P < .001 for WT vs KLF1+/E325K; +P < .05 and +++P < .001 for WT vs KLF1+/−). (F) Percentage of cells tested positive for anV with fluorescein isothiocyanate (FITC) and negative with propidium iodide (PI) during expansion (day 0) and on days 2, 4, 6, 9, and 11 of differentiation (mean ± SD; n = 3; ∗∗∗P < .001). (G) Western blot of WT, KLF1+/E325K, and KLF1+/− cells probed with antibody to cleaved caspase 3, and to β-actin as loading control (representative of 3 biological repeats). (H) Percentage of cells positive for anV using FITC and PI during expansion (day 0) and on days 2, 4, 6, 9, and 11 of differentiation (mean ± SD; n = 3; ∗∗∗P < .001). (I) Percentage of erythroid cell types during expansion (day 0) and on days 2, 4, 6, 8, and 10 of differentiation, analyzed after staining with May-Grünwald Giemsa (mean ± SD; n = 5; >100 cells counted per sample). (J) Expression of (i) BCAM, (ii) AQP1, and (iii) ICAM4 determined via quantitative polymerase chain reaction (qPCR; mean ± SD; n = 4; ∗P < .05; ∗∗∗P < .001), and abundance of (iv) CD44 protein determined via flow cytometry using an allophycocyanin (APC)-labeled anti-CD44 antibody (mean ± SD; n = 3; ∗P < .05; ∗∗∗P < .001). BasoE, basophilic erythroblast; Ortho, orthochromatic erythroblast; ProE, proerythroblast; Poly, polychromatic erythroblast; Retic, reticulocyte.
